The Role of Inflammatory Lipid Mediators on Colon Cancer Initiating Cells (CICs)

Colorectal cancer (CRC) is one of the major causes of cancer globally. Recent studies proposed a role for cancer initiating cells (CICs), a small subset of replication-competent cells, in colon carcinogenesis. Although the role of inflammatory lipid-mediators in CRC progression is well known, their role in the promotion of cancer-initiating cells remains to be elucidated. For this thesis, we investigated the role of eicosanoids – leukotriene D4 (LTD4) or prostaglandin E2 (PGE2) – on CIC properties and changes occurring in the tumor environment that could possibly support CIC-induced tumor growth. To this end, we identified the CICs on the basis of ALDH expression and evaluated their in vitro characteristics like colony formation, radio or chemoresistance and in vivo tumorigenic properties in the presence of LTD4 or PGE2. We showed that LTD4 and PGE2 enriched the ALDH+ cell population and augmented the colonies formation and tumor progression in xenograft mice model. The ALDH+ cells were also resistant to 5-fluorouracil and radiation that is additionally augmented by both the lipid-mediators. Moreover the impact of lipid inflammatory mediators on the stemness properties of CICs was evident by increased expression of genes that confer survival and self-renewal ability to CICs. In immunodeficient mice, LTD4 or PGE2 treatment amplified CIC-induced tumor growth. Furthermore, LTD4 and PGE2 increased cell proliferation activated β-catenin signaling and up-regulated COX-2. Additionally, LTD4 or PGE2 drive massive inflammatory responses identified as CD45+ enrichment, particularly of macrophages within tumors. The ability of ALDH+ cells to form tumors in immunodeficient mice could not be challenged by radiation therapy. In a separate series of experiments, we investigated the contribution of CICs in the development of sensitivity against montelukast, a CysLT1R antagonist. In this context we report that sensitivity of tumors against montelukast could depend on the variation in CICs content, activation of prosurvival factors such as BCL-2 and β-catenin signaling. Collectively, our data showed that LTD4 and PGE2 exacerbate CIC characteristics and promote tumor growth by allowing modifications in the tumor environment. New therapeutic strategies could aim to resolve not only cancer associated inflammation, but also to target CICs in order to achieve better remission and cure advanced colon cancer stages

The eicosanoids leukotriene D4 and prostaglandin E2 promote the tumorigenicity of colon cancer-initiating cells in a xenograft mouse model

Background: Colorectal cancer is one of the most common types of cancers worldwide. Recent studies have identified cancer-initiating cells (CICs) as a subgroup of replication-competent cells in the development of colorectal cancer. Although it is understood that an inflammation-rich tumor microenvironment presumably supports CIC functions, the contributory factors are not very well defined. The present study advances our understanding of the role of the eicosanoids leukotriene D4 (LTD4) and prostaglandin E2 (PGE2) in the tumorigenic ability of CICs and investigates the consequential changes occurring in the tumor environment that might support tumor growth. Methods: In this study we used human HCT-116 colon cancer ALDH+ cells in a nude mouse xenograft model. Protein expression and immune cell was determined in tumor-dispersed cells by flow cytometry and in tumor sections by immunohistochemistry. mRNA expressions were quantified using RT-q-PCR and plasma cytokine levels by Multiplex ELISA. Results: We observed that LTD4 and PGE2 treatment augmented CIC-induced tumor growth. LTD4-and PGE2-treated xenograft tumors revealed a robust increase in ALDH and Dclk1 protein expression, coupled with activated β-catenin signaling and COX-2 up-regulation. Furthermore, LTD4 or PGE2 accentuated the accumulation of CD45 expressing cells within xenograft tumors. Further analysis revealed that these infiltrating immune cells consisted of neutrophils (LY6G) and M2 type macrophages (CD206+). In addition, LTD4 and PGE2 treatment significantly elevated the plasma levels of cysteinyl leukotrienes and PGE2, as well as levels of IL-1β, IL-2, IL-6, TNF-α and CXCL1/KC/GRO. In addition, increased mRNA expression of IL-1β, IL-6 and IL-10 were detected in tumors from mice that had been treated with LTD4 or PGE2. Conclusion: Our data suggest that both LTD4 and PGE2 promote CICs in initiating tumor growth by allowing modifications in the tumor environment. Our data indicate that new therapeutic strategies targeting eicosanoids, specifically LTD4 and PGE2, could be tested for better therapeutic management of colon cancer

The WNT5A Agonist Foxy5 Reduces the Number of Colonic Cancer Stem Cells in a Xenograft Mouse Model of Human Colonic Cancer

BACKGROUND: The wingless-type mammary tumour virus integration site 5A (WNT5A) agonist Foxy5 was shown in vitro to affect intracellular signalling implicated in the regulation of colonic cancer stem cells (CSCs). MATERIALS AND METHODS: In order to study whether Foxy5 can modulate CSCs, either HT-29 or Caco-2 human colonic cancer cells, both lacking endogenous WNT5A expression, were inoculated subcutaneously into nude mice. RESULTS: Foxy5 reduced the expression of the stem-cell marker aldehyde dehydrogenase and, interestingly, the specific colon CSC marker double cortin-like kinase 1. Foxy5 also reduced active β-catenin and the expression of its downstream target Achaete Scute complex homolog 2, a CSC-preserving transcription factor. Foxy5 also reduced cyclo-oxygenase 2 expression, responsible for the formation of the CSC-promoting prostaglandin E2 (PGE2), but increased that of 15-hydroxyprostaglandin dehydrogenase expression, a PGE2-degrading enzyme. Accordingly, Foxy5 impairs both β-catenin and PGE2 signalling, both of which have been implicated in promoting the niche of colonic CSCs. CONCLUSION: Our data suggest that Foxy5 can complement the traditional adjuvant chemotherapeutic treatment to which CSCs are resistant

Cysteinyl leukotriene receptor 1 facilitates tumorigenesis in a mouse model of colitis-associated colon cancer

Cysteinyl leukotriene receptor 1 (CysLT1R) has been shown to be up-regulated in the adenocarcinomas of colorectal cancer patients, which is associated with a poor prognosis. In a spontaneous model of colon cancer, CysLT1R disruption was associated with a reduced tumor burden in double-mutant female mice (ApcMin/+/Cysltr1-/-) compared to ApcMin/+ littermates. In the current study, we utilized a genetic approach to investigate the effect of CysLT1R in the induced azoxymethane/dextran sulfate sodium (AOM/DSS) model of colitis-associated colon cancer. We found that AOM/DSS female mice with a global disruption of the Cysltr1 gene (Cysltr1-/-) had a higher relative body weight, a more normal weight/length colon ratio and smaller-sized colonic polyps compared to AOM/DSS wild-type counterparts. The Cysltr1-/- colonic polyps exhibited low-grade dysplasia, while wild-type polyps had an adenoma-like phenotype. The Cysltr1-/- colonic polyps exhibited significant decreases in nuclear β-catenin and COX-2 protein expression, while the normal crypts surrounding the polyps exhibited increased Mucin 2 expression. Furthermore, Cysltr1-/- mice exhibited an overall reduction in inflammation, with a significant decrease in proinflammatory cytokines, polyp 5-LOX expression and infiltration of CD45 leukocytes and F4/80 macrophages. In conclusion, the present genetic approach in an AOM/DSS model further supports an important role for CysLT1R in colon tumorigenesis

Montelukast, a CysLT1 receptor antagonist, reduces colon cancer stemness and tumor burden in a mouse xenograft model of human colon cancer

Inflammation is implicated in the etiology of sporadic colon cancer (CC), which is one of the leading causes of cancer-related deaths worldwide. Here, we report that inhibition of the inflammatory receptor CysLT1 through its antagonist, montelukast, is beneficial in minimizing stemness in CC and thereby minimizing tumor growth in a mouse xenograft model of human colon cancer. Upon treatment with montelukast, colonospheres derived from HT-29 and SW-480 human colon cancer cells exhibited a significant phenotypic change coupled with the downregulation of mRNA and protein expression of cancer stem cell (CSC) markers ALDH1 and DCLK1. Moreover, montelukast reduced the size of HT-29 cell-derived tumors in mice. The reduction in tumor size was associated with decreased levels of ALDH1A1, DCLK1, BCL2 mRNA and macrophage infiltration into the tumor tissue. Interestingly, this treatment elevated levels of the tumor suppressor 15-PGDH while reducing COX-2 expression. Our data highlight the association of CysLT1R with CSCs and demonstrate that inhibition of CysLT1R could prove beneficial in minimizing CSC-induced tumor growth. This work advances the notion that targeting CSCs is a promising approach to improve outcomes in those afflicted with colon cancer

Disrupting Inflammation-Associated CXCL8-CXCR1 Signaling Inhibits Tumorigenicity Initiated by Sporadic- and Colitis-Colon Cancer Stem Cells

Dysfunctional inflammatory pathways are associated with an increased risk of cancer, including colorectal cancer. We have previously identified and enriched for a self-renewing, colon cancer stem cell (CCSC) subpopulation in primary sporadic colorectal cancers (CRC) and a related subpopulation in ulcerative colitis (UC) patients defined by the stem cell marker, aldehyde dehydrogenase (ALDH). Subsequent work demonstrated that CCSC-initiated tumors are dependent on the inflammatory chemokine, CXCL8, a known inducer of tumor proliferation, angiogenesis and invasion. Here, we use RNA interference to target CXCL8 and its receptor, CXCR1, to establish the existence of a functional signaling pathway promoting tumor growth initiated by sporadic and colitis CCSCs. Knocking down either CXCL8 or CXCR1 had a dramatic effect on inhibiting both in vitro proliferation and angiogenesis. Likewise, tumorigenicity was significantly inhibited due to reduced levels of proliferation and angiogenesis. Decreased expression of cycle cell regulators cyclins D1 and B1 along with increased p21 levels suggested that the reduction in tumor growth is due to dysregulation of cell cycle progression. Therapeutically targeting the CXCL8-CXCR1 signaling pathway has the potential to block sustained tumorigenesis by inhibiting both CCSC- and pCCSC-induced proliferation and angiogenesis

Phosphatidylinositol 4,5-bisphosphate Directs Spermatid Cell Polarity and Exocyst Localization in Drosophila

This study identifies phosphoinositides as key regulators of spermatid cell polarity. Polarization and elongation of spermatids in Drosophila are regulated through local synthesis of PIP2 by Sktl, which drives polarized localization of the exocyst complex to promote targeted membrane delivery and polarization of the elongating spermatid cysts